PROJECT SUMMARY/ABSTRACT This project develops sperm molecular biomarkers to improve detection and monitoring of toxicant- induced testicular injury. Monitoring now relies upon semen parameters and serum hormone levels, which are insensitive and variable indicators of testicular toxicity. Sperm mRNA transcripts and DNA methylation marks are acquired during spermatogenesis and reflect the integrity of that process; therefore, measuring these sperm biomarkers provides insight into the testicular response to environmental and occupational exposures. In the past grant period, we characterized rat models of toxicant-induced testicular injury and identified rat sperm mRNA transcript alterations that are sensitive and specific indicators of testicular toxicity. We have also shown that human sperm mRNA transcripts and DNA methylation marks can be measured and are indicative of sperm dysfunction. In this grant period, additional rat models of toxicant exposure, including mixed exposures, will be evaluated (Specific Aim 1) to identify rat sperm mRNA transcripts and DNA methylation marks that reflect testicular toxicity (Specific Aim 2a). Building on these rat studies, we will collaborate with Project 3 to study trichloroethylene exposure, a key vapor intrusion indoor air contaminant (Specific Aim 2b). An optimized human-relevant sperm molecular biomarker panel will be developed by characterizing sperm mRNA and DNA methylation alterations in men receiving chronic methotrexate treatment in comparison to a control group of normal men (Specific Aim 3). This basic-to-clinical translational effort will be guided by the following working hypothesis: Sperm mRNA transcripts and DNA methylation marks are integrated molecular biomarkers of testicular injury that can be used to monitor low level mixed environmental exposures. The goal of this project is to apply novel sperm molecular biomarkers to well-defined animal models of testicular toxicity, enabling the translation of this technology for monitoring environmental and occupational exposures in humans. This goal will be achieved by fulfilling the following Specific Aims: Specific Aim 1: Evaluate the phenotypic and functional consequences of low level exposures to testicular toxicants that produce molecular alterations in rat sperm, including changes in testis histopathology, fertilizing capacity, and early embryogenesis. Specific Aim 2: a) Identify rat sperm mRNA transcripts and DNA methylation marks that are molecular biomarkers of testicular dysfunction following low-level exposure to cell type specific toxicants and mixtures of toxicants. b) In conjunction with Project 3, evaluate the male reproductive tract effects of trichloroethylene exposure in the rat with phenotypic and functional assays, and sperm biomarkers. Specific Aim 3: Translate these findings by examining sperm molecular biomarkers in an epidemiology study of normal men compared to men treated with methotrexate for autoimmune arthritis.